Streptavidin is an antibiotic produced by the bacteria Streptomyces avidinii and other Streptomyces species (E. O. Stapley et al., Antimicrobial Agents and Chemotherapy 1963, 20-27 (J. C. Sylvester ed. 1964)). It occurs naturally as a tetramer with a molecular weight of about 60,000 daltons. Streptavidin is characterized by its strong affinity for biotin and biotin derivatives and analogues. Each of the four identical subunits has a single biotin binding site (K. Hofmann et al., Proc. Natl. Acad. Sci. USA 77, 4666-68 (1980); L. Chaiet and F. J. Wolf, Archives of Biochemistry and Biophysics 106, 1-5 (1964)).
Because of its strong affinity for biotin, streptavidin has found widespread application, both commercially, and for applied and basic biomedical research, to study biotin-requiring enzymes and, when used in conjunction with biotinylated substances, to study the interactions between these substances and other products (K. Hofmann, supra; see also E. A. Bayer and M. Wilchek, Methods of Biochemical Analysis 26, 1-45 (1980); F. M. Finn et al., J. Biol Chem. 255, 5742-46 (1980)). Today, streptavidin is produced commercially by isolating it from the cell medium of Streptomyces avidinii. Purification of streptavidin from Streptomyces avidinii has resulted in very low yields of only 3-4 mg per liter of cell culture.
Recent advances in molecular biology have made it possible to produce large amounts of heterologous proteins in bacterial hosts. These include, for example, leukocyte interferon (S. Nagata et al., "Synthesis In E. coli Of A Polypeptide With Human Leukocyte Interferon Activity", Nature 284, 316-20 (1980)), antigens of human hepatitis B virus (C. J. Burrell et al., "Expression In Escherichia coli Of Hepatitis B Virus DNA Sequences Cloned In Plasmid pBR322," Nature 279, 43-47 (1979) and M. Pasek et al., "Hepatitis B Virus Genes And Their Expression In E. coli", Nature 282, 575-79 (1979)), SV40 t antigen (T. M. Roberts et al., "Synthesis Of Simian Virus 40 t Antigen In Escherichia coli", Proc. Natl. Acad. Sci. USA 76, 5596-5600 (1979)), and FMD viral antigens (H. Kupper et al., "Cloning Of cDNA Of Major Antigen Of Foot And Mouth Disease Virus And Expression In E. coli", Nature 289, 555-59 (1982)).
In general, these processes rely on the construction of recombinant DNA molecules characterized by a DNA sequence coding for the desired protein, polypeptide, peptide or amino acid operatively linked to an expression control sequence. Appropriate hosts are then transformed with these molecules to permit production of the desired product by fermentation. For DNA sequences, other than those prepared via chemical synthesis, the construction of such recombinant DNA molecules often comprises the-steps of producing a single-stranded DNA copy ("cDNA") of a messenger RNA ("mRNA") template for the desired product; converting the CDNA to double-stranded DNA and operatively linking the DNA to an appropriate expression control sequence in an appropriate cloning vehicle. The recombinant DNA molecule is then employed to transform an appropriate host. Such transformation may permit the host to produce the desired product when it is fermented under appropriate conditions.
A further improvement of the above technology has made it possible to excrete the selected protein, polypeptide, peptide or amino acid through the membrane of the host cell as it is produced by:
forming a hybrid gene consisting of a DNA sequence from an extracellular or periplasmic carrier protein that is excreted by the host, and a heterologous DNA fragment which codes for the selected protein, polypeptide or amino acid;
transforming the host with that hybrid gene operatively linked to an expression control sequence; and
culturing the transformed host to synthesize and to secrete the selected protein, polypeptide, peptide or amino acid.
Such techniques are disclosed, for example, by L. Villa-Komaroff et al., "A Bacterial Clone Synthesizing Pro-Insulin," Proc. Natl. Acad. Sci. USA 75, 3727-31 (1978), and U.S. Pat. No. 4,411,994. However, any protein, polypeptide, peptide or amino acid made by this method, although separated from intracellular proteins and cell debris by secretion, must still be recovered from the cell medium or periplasmic space. This recovery generally involves a purification scheme that is less effective and less simple than desired. It also generally results in product losses.